At least three molybdenum-containing enzymes, sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase are known to be present in human tissues. The severe pathological symptoms observed in patients with simple sulfite oxidase deficiency or with combined deficiency of all Mo-containing enzymes attests to the essentiality of Mo for normal health and mental development in humans. The basis for the combined deficiency of Mo enzymes is the presence of a common cofactor in all Mo enzymes, from all organisms spanning the phylogenetic scale, in which the metal plays a biochemical role. Recently this laboratory has shown that the Mo cofactor contains a novel pterin, and that the same pterin is present in every molybdenum enzyme examined, but not in several non-molybdo proteins tested. The aim of this proposal is to carry out detailed studies on several aspects of the moly bdenum cofactor. The chemical structures of two major inactive derivatives of the cofactor, as well as of the active cofactor itself, will be elucidated. The pathway of biosynthesis of the cofactor in Escherichia coli will be established, using isotope trace methods and by isolating and characterizing cofactor mutants. The question of whether the cofactor is synthesized in animals or is a nutritional component, i.e., a vitamin, will be examined both by nutritional methods and by in vivo and in vitro isotopic techniques. Using a previously devloped model for experimentl Mo deficiency in rats the causative factor for the pathophysiology of cofactor deficiency will be explored - the possibilities being, on the one hand, toxicity of SO3 and secondary metabolities arising from the reaction of SO3 with other molecules, and on the other hand, a deficiency of SO4 arising from the absence of sulfite oxidase activity. Because of the unexpected absence of sulfite oxidase CRM in patients with Mo cofactor deficiency it is also proposed to carry out studies on the in vitro synthesis of the sulfite oxidase protein, the ultimate aim being to determine why Mo cofactor deficiency leads to a loss of the CRM of one of the molybdenum-containing proteins.